EP2676679B1

EP2676679B1 - Formulations which stabilize and inhibit precipitation of immunogenic compositions

Info

Publication number: EP2676679B1
Application number: EP13185292.3A
Authority: EP
Inventors: Lakshmi Khandke; Hanyoung Han; Robert Chancery Seid Jr.; Zhaowei Jin; Jee Loon Look; Ronald Malone; Xudong Yang; Ying Chen
Original assignee: Wyeth LLC
Current assignee: Wyeth LLC
Priority date: 2006-04-26
Filing date: 2007-04-19
Publication date: 2019-01-02
Anticipated expiration: 2027-04-19
Also published as: CA2650056C; BRPI0710918B8; CN105879021B; JP2014221806A; SI2679244T1; ES2712956T3; PT2679244T; DK2679245T3; LT2676679T; PT2676679T; DK2010219T3; DK2676679T3; KR20210111326A; CN103768615A; KR20170122857A; CA2873346A1; SI2010219T1; CL2007001185A1; ZA200809167B; KR20200032268A

Description

FIELD OF THE INVENTION

The present invention generally relates to the fields of immunology, bacteriology, vaccine formulation, protein stability and process development. More particularly, the invention relates to novel formulations which inhibit precipitation of immunogenic compositions.

BACKGROUND OF THE INVENTION

It is generally accepted in the bio-pharmaceutical arts, that improving the stability of an immunogenic composition (e.g., a protein immunogen, a polysaccharide-protein conjugate) is a necessary and highly desirable goal. For example, an immunogenic composition must appear fresh, elegant and professional when administered to a patient. Any changes in stability and/or physical appearance of the immunogenic composition, such as color change, clouding or haziness, may cause a patient or consumer to lose confidence in the product. Furthermore, because many immunogenic formulations are dispensed in multiple-dose containers, uniformity of dose content of the active ingredient (e.g., a polysaccharide-protein conjugate) over time must be assured (e.g., a cloudy solution can lead to a nonuniform dosage pattern). Additionally, the immunogenic composition must be active throughout its "expected" shelf life, wherein any breakdown of the immunogenic composition to an inactive or otherwise undesired form (e.g., an aggregate) lowers the total concentration of the product.
Several reports in the literature have suggested that the stability of a particular immunogenic composition (e.g., a protein immunogen, a polysaccharide-protein conjugate) is at least in part dependent upon the specific protein or carrier protein (Ho et al., 2001; Ho et al., 2002; Bolgiano et al., 2001). For example, stability analysis of meningococcal C (MenC) polysaccharides and Haemophilus influenzae type b (Hib) polysaccharides, conjugated to either a tetanus toxoid (TT) or a CRM₁₉₇ carrier protein, revealed different stability profiles dependent on the carrier protein (Ho et al., 2002). In another study (Ho et al., 2001), MenC-CRM₁₉₇ conjugates from two different manufacturers were analyzed (Ho et al., 2001), wherein the MenC-CRM₁₉₇ conjugates differed in their conjugation chemistry and length of conjugate polysaccharide (both having the same carrier protein, CRM₁₉₇). Data from this study further indicated that factors such as conjugation chemistry (e.g., reductive amination either directly or via a chemical spacer group), number of conjugation sites, polysaccharide chain length, pH, storage buffer, storage temperature(s) and freeze/thaw cycles also influence the stability of an immunogenic composition.
Thus, when developing a formulation for an immunogenic composition, many factors must be considered to ensure a safe, stable, robust and cost effective product. Such considerations include, but are not limited to, chemical stability of the immunogenic composition (e.g., hydrolysis of saccharides, de-polymerization of polysaccharides, proteolysis or fragmentation of proteins), physical/thermal stability of the immunogenic composition (e.g., aggregation, precipitation, adsorption), compatibility of the immunogenic composition with the container/closure system, interactions between immunogenic composition and inactive ingredients (e.g., buffers, salts, excipients, cryoprotectants), the manufacturing process, the dosage form (e.g., lyophilized, liquid), the environmental conditions encountered during shipping, storage and handling (e.g., temperature, humidity, shear forces), and the length of time between manufacture and usage.
It has been suggested in the art, that silicone oil, which induces protein secondary and tertiary conformational changes, might be responsible for the aggregation/precipitation seen in certain protein pharmaceutical preparations (Jones et al., 2005). For example, several reports in the 1980s implicated the release of silicone oil from disposable plastic syringes as the causative agent in the aggregation of human insulin (Chantelau and Berger, 1985; Chantelau et al., 1986; Chantelau, 1989; Bernstein, 1987; Baldwin, 1988; Collier and Dawson, 1985). Chantelau et al. (1986) observed that after three or more withdrawals from a ten-dose preparation of insulin (using a siliconized disposable syringe), the vial would begin clouding due silicone oil contamination, thereby resulting in aggregation and deactivation of the insulin (Chantelau et al., 1986). Paradoxically, silicone oil is a necessary component of plastic syringes, as it serves to lubricate the rubber plunger and facilitate transfer of the plunger down the syringe barrel (i.e., silicone oil improves the syringeability of the formulation).
Furthermore, the use of silicone oil is not limited to syringes, as it is used as a coating for glass vials to minimize protein adsorption, as a lubricant to prevent conglomeration of rubber stoppers during filing procedures, as a lubricant critical to the processability/machinability of glass and elastomeric closures and as a lubricant to ease needle penetration of vial rubber stoppers. Additionally, the siliconization of syringes, glass vials, rubber stoppers and the like, is not a well controlled nor standardized process, and as such, there is a high degree of variability of the silicone oil content from one lot to another.
EP2425856 (prior art under Art. 54(3) EPC) discloses a multivalent immunogenic composition comprising 13 distinct polysaccharide-protein conjugates.
WO 2007/060548 (prior art under Art. 54(3) EPC) discloses hybrid and tandem proteins of a protein from Neisseria meningitidis named NMB 1870.
WO 01/41800 discloses compositions and methods for the preparation of MenC-CRM₁₉₇ conjugate.
US 2004/047882 discloses a two component vaccine containing a first vaccine, adjuvanted with an oil-in-water emulsion comprising 5% squalene, 0.5% polysorbate 80 and 0.5% sorbitan trioleate in aqueous citrate buffer pH 6.5, and a nonadjuvated second vaccine as combination partners for the simultaneous, separate or phased application for immunization against viral, bacterial or parasitic infectious diseases.
WO 02/05846 discloses a quadrivalent combination vaccine comprising diphtheria toxoid, tetanus toxoid, whole cell pertussis, and HBsAg, and a method for preparing the same. In the preparation of the DTwPH combination vaccine, diphtheria toxoid and tetanus toxoid are adsorbed onto aluminum phosphate (AlPO4) gels, and HBsAg is adsorbed onto aluminum hydroxide (Al(OH)3) gel.
There is therefore an ongoing need in the art for formulations which enhance stability and inhibit precipitation of immunogenic compositions.

SUMMARY OF THE INVENTION

The present invention broadly relates to formulations which stabilize and inhibit precipitation of immunogenic compositions. More specifically in certain embodiments, the present invention is directed to formulations which inhibit precipitation of immunogenic compositions comprised in container means. In one specific embodiment, the invention is directed to formulations which stabilize immunogenic compositions against silicone oil interactions, shear forces and shipping agitation.
Thus, in certain aspects, the disclosure is directed to formulations which stabilize a polysaccharide-protein conjugate, the formulation comprising (i) a pH buffered saline solution, wherein the buffer has a pKa of about 3.5 to about 7.5, (ii) a surfactant and (iii) one or more polysaccharide-protein conjugates. In one specific aspect, the polysaccharide-protein conjugate formulation is comprised in a container means. In certain aspects, the container means is selected from one or more of the group consisting of a vial, a vial stopper, a vial closure, a glass closure, a rubber closure, a plastic closure, a syringe, a syringe stopper, a syringe plunger, a flask, a beaker, a graduated cylinder, a fermentor, a bioreactor, tubing, a pipe, a bag, a jar, an ampoule, a cartridge and a disposable pen. In certain aspects, the container means is siliconized.
In certain aspects, the pH buffered saline solution of the formulations has a pH of 5.5 to 7.5. In other aspects, the buffer is phosphate, succinate, histidine or citrate. In certain aspects, the buffer is succinate at a final concentration of 1 mM to 10 mM and pH 5.8 to 6.0. In one particular aspect, the final concentration of the succinate buffer is 5 mM. In other aspects, the salt in the pH buffered saline solution comprises magnesium chloride, potassium chloride, sodium chloride or a combination thereof. In one particular aspect, the salt in the pH buffered saline solution is sodium chloride.
In another aspect, the surfactant of the formulations is selected from the group consisting of polysorbate 20 (Tween™20), polysorbate 40 (Tween™40), polysorbate 60 (Tween™60), polysorbate 65 (Tween™65), polysorbate 80 (Tween™80), polysorbate 85 (Tween™85), Triton™ N-101, Triton™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), polyoxyethylene-35-ricinoleate (Cremophor EL™), soy lecithin and a poloxamer. In one particular aspect, the surfactant is polysorbate 80. In another aspect, the final concentration of the polysorbate 80 in formulation is at least 0.01% to 10% polysorbate 80 weight/volume of the formulation. In other aspects, the final concentration of the polysorbate 80 in the formulation is 0.01% polysorbate 80 weight/volume of the formulation. In yet other aspects, the final concentration of the polysorbate 80 in the formulation is 0.05% polysorbate 80 weight/volume of the formulation. In another aspect, the final concentration of the polysorbate 80 in the formulation is 0.1% polysorbate 80 weight/volume of the formulation. In certain other aspects, the final concentration of the polysorbate 80 in the formulation is 1.0% polysorbate 80 weight/volume of the formulation. In yet other aspects, the final concentration of the polysorbate 80 in the formulation is 10.0% polysorbate 80 weight/volume of the formulation.
In another aspect, the polysaccharide-protein conjugate comprises one or more pneumococcal polysaccharides. In certain embodiments, the one or more pneumococcal polysaccharides are a S. pneumoniae serotype 4 polysaccharide, a S. pneumoniae serotype 6B polysaccharide, a S. pneumoniae serotype 9V polysaccharide, a S. pneumoniae serotype 14 polysaccharide, a S. pneumoniae serotype 18C polysaccharide, a S. pneumoniae serotype 19F polysaccharide, a S. pneumoniae serotype 23F polysaccharide, a S. pneumoniae serotype 1 polysaccharide, a S. pneumoniae serotype 3 polysaccharide, a S. pneumoniae serotype 5 polysaccharide, a S. pneumoniae serotype 6A polysaccharide, a S. pneumoniae serotype 7F polysaccharide and a S. pneumoniae serotype 19A polysaccharide. In certain embodiments, the protein of the polysaccharide-protein conjugate formulation is selected from the group consisting of CRM₁₉₇, a tetanus toxoid, a cholera toxoid, a pertussis toxoid, an E. coli heat labile toxoid (LT),a pneumolysin toxoid, pneumococcal surface protein A (PspA), pneumococcal adhesin protein A (PsaA), a C5a peptidase from Streptococcus, Haemophilus influenzae protein D, ovalbumin, keyhole limpet haemocyanin (KLH), bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD).
In one specific aspect, the polysaccharide-protein conjugate formulation is a 7-valent pneumococcal conjugate (7vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide.
In another specific aspect, the polysaccharide-protein conjugate formulation is a 13-valent pneumococcal conjugate (13vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S . pneumoniae serotype 1 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 3 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 5 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6A polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 7F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 19A polysaccharide conjugated to a CRM₁₉₇ polypeptide.
In other aspects, the formulation further comprises one or more meningococcal polysaccharides, one or more meningococcal antigenic proteins, or a combination thereof. In yet other aspects, the formulation further comprises one or more streptococcal polysaccharides, one or more streptococcal antigenic proteins, or a combination thereof.
In certain other aspects, the formulation further comprises one or more adjuvants. Exemplary suitable adjuvants are described herein below.
In another embodiment, the invention is directed to formulations which inhibit silicone induced precipitation of a polysaccharide-protein conjugate comprised in a siliconized container means, the formulation comprising (i) a pH buffered saline solution, wherein the buffer has a pKa of about 3.5 to about 7.5, (ii) an aluminum salt and (iii) one or more polysaccharide-protein conjugates. In certain embodiments, the siliconized container means is selected from one or more of the group consisting of a vial, a vial stopper, a vial closure, a glass closure, a rubber closure, a plastic closure, a syringe, a syringe stopper, a syringe plunger, a flask, a beaker, a graduated cylinder, a fermentor, a bioreactor, tubing, a pipe, a bag, a jar, an ampoule, a cartridge and a disposable pen.
In certain embodiments, the pH buffered saline solution in the formulations has a pH of 5.5 to 7.5. In other embodiments, the buffer in the formulations is phosphate, succinate, histidine or citrate. In yet other embodiments, the buffer is succinate at a final concentration of 1 mM to 10 mM and pH 5.8 to 6.0. In one particular embodiment, the final concentration of the succinate buffer is 5 mM. In still other embodiments, the salt in the pH buffered saline solution comprises magnesium chloride, potassium chloride, sodium chloride or a combination thereof. In one particular embodiment, the salt in the pH buffered saline solution is sodium chloride.
In other embodiments, the aluminum salt is aluminum hydroxide, aluminum phosphate or aluminum sulfate. In one specific embodiment, the aluminum salt is aluminum phosphate.
In certain other embodiments, the formulation further comprises polysorbate 80 (Tween™80). In one specific embodiment, the final concentration of the polysorbate 80 in formulation is at least 0.01% to 10% polysorbate 80 weight/volume of the formulation.
In another embodiment, the polysaccharide-protein conjugate comprises one or more pneumococcal polysaccharides. In certain embodiments, the one or more pneumococcal polysaccharides are a S. pneumoniae serotype 4 polysaccharide, a S. pneumoniae serotype 6B polysaccharide, a S. pneumoniae serotype 9V polysaccharide, a S. pneumoniae serotype 14 polysaccharide, a S. pneumoniae serotype 18C polysaccharide, a S. pneumoniae serotype 19F polysaccharide, a S. pneumoniae serotype 23F polysaccharide, a S. pneumoniae serotype 1 polysaccharide, a S. pneumoniae serotype 3 polysaccharide, a S. pneumoniae serotype 5 polysaccharide, a S. pneumoniae serotype 6A polysaccharide, a S. pneumoniae serotype 7F polysaccharide and a S. pneumoniae serotype 19A polysaccharide.
In certain other embodiments, the protein of the polysaccharide-protein conjugate formulation is selected from the group consisting of CRM₁₉₇, a tetanus toxoid, a cholera toxoid, a pertussis toxoid, an E. coli heat labile toxoid (LT),a pneumolysin toxoid, pneumococcal surface protein A (PspA), pneumococcal adhesin protein A (PsaA), a C5a peptidase from Streptococcus, Haemophilus influenzae protein D, ovalbumin, keyhole limpet haemocyanin (KLH), bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD).
In one particular embodiment, the polysaccharide-protein conjugate formulation is a 7-valent pneumococcal conjugate (7vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide.
In another specific embodiment, the polysaccharide-protein conjugate formulation is a 13-valent pneumococcal conjugate (13vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 1 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 3 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 5 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6A polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 7F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 19A polysaccharide conjugated to a CRM₁₉₇ polypeptide.
In yet other aspects, the formulation further comprises one or more meningococcal polysaccharides, one or more meningococcal antigenic proteins, or a combination thereof.
In another aspect, the formulation further comprises one or more streptococcal polysaccharides, one or more streptococcal antigenic proteins, or a combination thereof.
In certain other embodiments, the formulation further comprises one or more adjuvants. Exemplary suitable adjuvants are described herein below.
The present invention is defined by the appended claims. Subject matters which are not encompassed by the scope of the claims do not form part of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Reference

Figure 1 shows the stability of Streptococcal C5a peptidase (SCP) formulations (filled in syringes) before and after two days of gentle agitation (60 cpm) on a horizontal orbital shaker. The data presented in FIG. 1A is the two day stability of the SCP formulated without any Tween™80 (i.e., 0%), whereas the data in FIG. 1B is the two day stability of the SCP formulated with 0.025% Tween™80. The buffers used in the formulations shown in FIG. 1A and 1B are succinate buffered saline (SBS), phosphate buffered saline (PBS) and tris(hydroxymethyl)aminomethane (TRIS).

Figure 2 shows the total antigenicity loss of the 13vPnC formulated with AlPO₄ (0.25 mg/ml) and filled in a BD Hypak syringe, after two hours, eight hours and twenty-four hours of agitation at 500 rpm and 2-8°C.
Figure 3 shows the total antigenicity loss of the 13vPnC formulated with AlPO₄ (0.25 mg/ml) and filled in an un-siliconized syringe, after two hours, eight hours and twenty-four hours of agitation at 500 rpm and 2-8°C.
Figure 4 shows the total antigenicity loss of the 13vPnC formulated with AlPO₄ (0.25 mg/ml) and filled in a Vetter syringe, after two hours, eight hours and twenty-four hours of agitation at 500 rpm and 2-8°C.
Figure 5 shows the total antigenicity loss of the 13vPnC formulated with AlPO₄ (0.25 mg/ml) and filled in a Schott TopPac syringe, after two hours, eight hours and twenty-four hours of agitation at 500 rpm and 2-8°C.
Figure 6 shows the total antigenicity loss of the 13vPnC formulated with (FIG. 6A) and without (FIG. 6B) AlPO₄ (0.25 mg/ml) and filled in a BD Baked syringe, after two hours, eight hours and twenty-four hours of agitation at 500 rpm and 2-8°C.
Figure 7 shows the total antigenicity loss of the 13vPnC formulated with (FIG. 7A) and without (FIG. 7B) AlPO₄ (0.25 mg/ml) and filled in a BünderGlas PS2 syringe, after two hours, eight hours and twenty-four hours of agitation at 500 rpm and 2-8°C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention addresses an ongoing need in the art to improve the stability of immunogenic compositions such as polysaccharide-protein conjugates and protein immunogens. Thus, the present invention broadly relates to aluminum salt formulations which stabilize and inhibit precipitation of immunogenic compositions. More particularly, the invention described hereinafter, addresses a need in the art for formulations which stabilize and inhibit particulate formation (e.g., aggregation, precipitation) of immunogenic compositions which are processed, developed, formulated, manufactured and/or stored in siliconized container means such as fermentors, bioreactors, vials, flasks, bags, syringes, rubber stoppers, and tubing.
As set forth above in the Background of the Invention, various factors influence the stability of immunogenic compositions, including chemical stability of the immunogenic composition, physical/thermal stability of the immunogenic composition, compatibility of the immunogenic composition with the container/closure system, interactions between immunogenic composition and inactive ingredients (e.g., buffers, salts, excipients, cryoprotectants), manufacturing processes, dosage form, environmental conditions encountered during shipping, storage and handling (e.g., temperature, humidity, shear forces), and the length of time between manufacture and usage.
The stability of an immunogenic composition of the invention is readily determined using standard techniques, which are well known and routine to those of skill in the art. For example, an immunogenic composition is assayed for stability, aggregation, immunogenicity, particulate formation, protein (concentration) loss and methods including light scattering, optical density, sedimentation velocity centrifugation, sedimentation equilibrium centrifugation, circular dichroism (CD), Lowry assay, bicinchoninic acid (BCA) assay and antibody binding.
As set forth in detail herein, the present disclosure relates to the unexpected and surprising results that formulating an immunogenic composition with a surfactant such as Tween™80 significantly enhances the stability and inhibits precipitation of an immunogenic composition. For example, it was observed in the present disclosure (e.g., see Example 2), that a thirteen-valent pneumococcal conjugate (13vPnC), formulated in buffered saline and filled in a single does syringe, would begin precipitating out of solution within ten minutes at 2-8°C upon gentle agitation via a horizontal orbital shaker. (The horizontal orbital shaker was used to simulate typical process, shipping and storage conditions of a 13vPnC immunogenic composition). However, it was surprisingly observed that the 13vPnC, formulated in buffered saline and 0.001% Tween™80, filled in a single dose syringe and gently agitated at 2-8°C, was stable for twenty-five days with no visible signs of precipitation (data not shown). Thus, this data demonstrated that the addition of a surfactant (e.g., Tween™80) to an immunogenic composition formulation enhances the stability of the immunogenic composition.
A second stability study of the 13vPnC further confirmed that the addition of a surfactant to the formulation significantly enhanced the stability of the 13vPnC. For example, the stability (i.e., assayed by measuring change in 13vPnC antigenicity) of a 13vPnC formulation with 0.05% Tween™80 (Table 1) and without Tween™80 (0.0%, Table 1) was assessed over a two hour time period. As is shown in Table 1, there was a significant decrease in antigenicity of the thirteen serotype polysaccharides (formulated without Tween™80) within the two hour assay . Quite dramatically however, the 13vPnC formulation comprising 0.05% Tween™80 (Table 1), demonstrated robust stability throughout the two hour antigenicity assay. It was also observed that the 13vPnC formulated in 250 mL glass bottles with either 0.01% Tween™80 or 0.05% Tween™80 could withstand significant shear forces induced via vortexing the formulations for thirty minutes at 2-8°C, with little or no loss in antigenicity (e.g., see Example 2, Table 2).
In other experiments (Example 3), it was demonstrated that the stability of an immunogenic streptococcal C5a peptidase (SCP) composition was greatly enhanced when formulated with a surfactant such as Tween™80. For example, as shown in FIG. 1A, after two days of vortexing an SCP (55 µg/mL) formulated in either a 5 mM succinate buffer (pH 6.0), a 10 mM phosphate buffer (pH 7.0 and 7.4) or a 10 mM Tris buffer (pH 7.5), there was a significant decrease (e.g., greater than 90%) in the SCP concentration. However, as shown in FIG. 1B, the addition of 0.025% Tween™80 to the SCP succinate, SCP phosphate and SCP Tris formulations, prior to vortexing for two days, completely inhibited the SCP loss which was observed in FIG. 1A.
A 13vPnC immunogenic composition of the invention may also be formulated with or without an adjuvant, such as aluminum phosphate (AlPO₄). Thus, in a separate series of experiments (Example 4), 13vPnC immunogenic compositions were formulated in 5 mM succinate buffer (pH 5.8), 0.85% NaCl and AlPO₄ (0.25 mg aluminum/ml), without the addition of a surfactant (e.g., no Tween™80 was included in the formulation).
In these experiments, the 13vPnC immunogenic composition (formulated in the presence of AlPO₄) were filled in various siliconized and non-siliconized container means (e.g., see Table 3) and subjected to simulated shipping and handling conditions via agitation at 2-8°C. It was observed in these experiments (Example 4), that the container means with higher silicone content exhibited a higher degree of 13vPnC particulate formation and a higher percent of 13vPnC antigenicity loss. An FTIR analysis of the particulates indicated that the particulates consisted of protein and silicone (data not shown) and that about 85% of the 13vPnC is bound to the AlPO₄, wherein the remaining 15% was free (not bound to AlPO₄) 13vPnC in solution.
In another experiment comparing 13vPnC immunogenic compositions formulated with and without AlPO₄, which were then filled in identical syringes, it was observed that the 13vPnC formulated without AlPO₄ sustained greater antigenicity losses than 13vPnC with AlPO₄ in the syringes tested (e.g., see FIG. 6 and FIG. 7).
Thus, the invention as set forth herein, is directed to novel formulations which stabilize and inhibit aggregation or precipitation of immunogenic compositions polysaccharide-protein conjugates (e.g., a 13vPnC), against the various factors which influence the stability of immunogenic compositions (e.g., shear forces, shipping agitation, silicone oil interactions, adsorption, manufacturing processes, temperature, humidity, length of time between manufacture and usage, etc.).
In certain embodiments, the invention is directed to a formulation which inhibits silicone induced precipitation of a polysaccharide-protein conjugate comprised in a siliconized container means, the formulation comprising a pH buffered saline solution, wherein the buffer has a pKa of about 3.5 to about 7.5, an aluminum salt and one or more polysaccharide-protein conjugates wherein the polysaccharide-protein conjugate comprises one or more pneumococcal polysaccharides. As defined hereinafter, the terms "precipitation", "precipitate" "particulate formation", "clouding" and "aggregation" may be used interchangeably and are meant to refer to any physical interaction or chemical reaction which results in the "aggregation" of a polysaccharide-protein conjugate or a protein (or polypeptide) immunogen. The process of aggregation (e.g., protein aggregation) is well known (but not well understood) and described in the art, and is often influenced by numerous physicochemical stresses, including heat, pressure, pH, agitation, shear forces, freeze-thawing, dehydration, heavy metals, phenolic compounds, silicon oil and denaturants.
As defined hereinafter, a "polysaccharide-protein conjugate", a "pneumococcal conjugate", a "7-valent pneumococcal conjugate (7vPnC)", a "13-valent pneumococcal conjugate (13vPnC)", a "streptococcal C5a peptidase (SCP) immunogenic composition" and a "N. meningitidis 2086 protein immunogenic composition" includes liquid formulations, frozen liquid formulations and solid (e.g., freeze-dried or lyophilized) formulations.

A. SURFACTANTS

As set forth above, the invention is directed to formulations which stabilize and inhibit aggregation of immunogenic compositions against the various factors which influence the stability of immunogenic compositions (e.g., shear forces, shipping agitation, silicone oil interactions, adsorption, manufacturing processes, temperature, humidity, length of time between manufacture and usage, etc.). In certain embodiments, the invention is directed to formulations comprising a surfactant.
A surfactant (or a surface-active agent) is generally defined as (a) a molecule or compound comprising a hydrophilic group or moiety and a lipophilic (hydrophobic) group or moiety and/or (b) a molecule, substance or compound that lowers or reduces surface tension of a solution. As defined herein, a "surfactant" of the present invention is any molecule or compound that lowers the surface tension of an immunogenic composition formulation.
A surfactant used in a formulation can comprise any surfactant or any combination of surfactants which stabilizes and inhibits aggregation of an immunogenic composition described herein. Thus, a surfactant includes polysorbate 20 (Tween™20), polysorbate 40 (Tween™40), polysorbate 60 (Tween™60), polysorbate 65 (Tween™65), polysorbate 80 (Tween™80), polysorbate 85 (Tween™85), Triton™ N-101, Triton™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), polyoxyethylene-35-ricinoleate (Cremophor EL™), soy lecithin, poloxamer, hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide, methoxyhexadecylgylcerol, pluronic polyols, polyamines (e.g., pyran, dextransulfate, poly IC, carbopol), peptides (e.g., muramyl peptide and dipeptide, dimethylglycine, tuftsin), oil emulsions, mineral gels (e.g., aluminum phosphate) and immune stimulating complexes (ISCOMS).
A person of skill in the art may readily determine a suitable surfactant or surfactant combination by measuring the surface tension of a particular immunogenic composition formulation in the presence and absence of the surfactant(s). Alternatively, a surfactant is evaluated qualitatively (e.g., visual inspection of particulate formation) or quantitatively (e.g., light scattering, sedimentation velocity centrifugation, optical density, antigenicity) for its ability to reduce, inhibit or prevent aggregation of an immunogenic composition.

B. CONTAINER MEANS

The invention is directed to formulations of immunogenic compositions comprised in a container means. As defined herein, a "container means" of the present invention includes any composition of matter which is used to "contain", "hold", "mix", "blend", "dispense", "inject", "transfer", "nebulize", etc. an immunogenic composition during research, processing, development, formulation, manufacture, storage and/or administration. For example, a container means of the present invention includes, but is not limited to, general laboratory glassware, flasks, beakers, graduated cylinders, fermentors, bioreactors, tubings, pipes, bags, jars, vials, vial closures (e.g., a rubber stopper, a screw on cap), ampoules, syringes, syringe stoppers, syringe plungers, rubber closures, plastic closures and glass closures. A container means of the present invention is not limited by material of manufacture, and includes materials such as glass, metals (e.g., steel, stainless steel, aluminum, etc.) and polymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers).
The skilled artisan will appreciate that the container means set forth above are by no means an exhaustive list, but merely serve as guidance to the artisan with respect to the variety of container means which are used to contain, hold, mix, blend, dispense, inject, transfer, nebulize, etc. an immunogen or immunogenic composition during research, processing, development, formulation, manufacture, storage and/or administration of the composition. Additional container means contemplated for use in the present invention may be found in published catalogues from laboratory equipment vendors and manufacturers such as United States Plastic Corp. (Lima, OH), VWR™ (West Chester, PA), BD Biosciences (Franklin Lakes, NJ), Fisher Scientific International Inc. (Hampton, NH) and Sigma-Aldrich (St. Louis, MO).
Thus, the novel formulations of the present invention are particularly advantageous in that they stabilize and inhibit precipitation of immunogenic formulations comprised in a container means throughout the various stages of research, processing, development, formulation, manufacture, storage and/or administration of the composition. The novel formulations of the invention not only stabilize immunogenic compositions against physical/thermal stresses (e.g., temperature, humidity, shear forces, etc.), they also enhance stability and inhibit precipitation of immunogenic compositions against negative factors or influences such as incompatibility of the immunogenic composition with the container/closure system (e.g., a siliconized container means).
Thus, the formulations of the present invention are particularly useful in stabilizing the immunogen (i.e., a polysaccharide-protein conjugate) against the silicon oil induced precipitation and precipitation described above. For example, co-pending U.S. Application No. 60/795,098, filed April 26, 2006 , describes the aggregation of immunogenic compositions in the presence silicon oil found on container means such syringes, glass vials, rubbers stoppers and the like, wherein the addition of a surfactant to the container means prevented the silicon oil induced aggregation of these immunogenic compositions.

C. ADJUVANTS AND PHARMACEUTICAL CARRIERS/EXCIPIENTS

In certain embodiments, the immunogenic compositions of the invention are further formulated with an adjuvant. An adjuvant is a substance that enhances the immune response when administered together with an immunogen or antigen. A number of cytokines or lymphokines have been shown to have immune modulating activity, and thus may be used as adjuvants, including the interleukins 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Patent No. 5,723,127 ), 13, 14, 15, 16, 17 and 18 (and its mutant forms), the interferons-α, β and γ, granulocyte-macrophage colony stimulating factor (GMCSF, see, e.g., U.S. Patent No. 5,078,996 and ATCC Accession Number 39900), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor (GCSF), and the tumor necrosis factors α and β (TNF). Still other adjuvants useful in this invention include chemokines MCP-1, MIP-1α, MIP-1β, and RANTES.
In certain embodiments, an adjuvant used to enhance an immune response of an immunogenic composition formulation includes MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094 . Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in United States Patent No. 6,113,918 . One such AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529). This 529 adjuvant is formulated as an aqueous form or as a stable emulsion (RC529-SE).
Still other adjuvants include mineral oil and water emulsions, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate etc., Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic polyols, muramyl dipeptide, killed Bordetella, saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, MA.), described in U.S. Patent No. 5,057,540 , and particles generated therefrom such as ISCOMS (immunostimulating complexes), ISCOMATRIX (CSL Limited, Parkville, Australia), described in U.S. Patent No. 5,254,339 , Mycobacterium tuberculosis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif ( U.S. Patent No. 6,207,646 ), IC-31 (Intercell AG, Vienna, Austria), described in European Patent Nos. 1,296,713 and 1,326,634 , a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302 and WO 92/19265 .
Also useful as adjuvants (and carrier proteins) are cholera toxins and mutants thereof, including those described in published International Patent Application number WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid (other than aspartic acid), preferably a histidine). Similar CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid). Other CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid positions 35 and 36).
In certain embodiments, the immunogenic composition formulations comprise a pharmaceutically acceptable diluent, excipient or a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable diluent is sterile water, water for injection, sterile isotonic saline or a biological buffer. The polysaccharide-protein conjugates and/or protein immunogens are mixed with such diluents or carriers in a conventional manner. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents compatible with administration to humans or other vertebrate hosts. The appropriate carrier is evident to those skilled in the art and will depend in large part upon the route of administration.
For example, excipients that may be present in the immunogenic composition formulation are preservatives, chemical stabilizers and suspending or dispersing agents. Typically, stabilizers and preservatives are optimized to determine the best formulation for efficacy in the targeted recipient (e.g., a human subject). Examples of preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Examples of stabilizing ingredients include casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried milk.
In certain embodiments, an immunogenic composition formulation is prepared for administration to human subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories. Thus, the immunogenic composition formulations may also include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.

D. IMMUNOGENS

A polysaccharide-protein conjugate formulation of the invention comprises one or more pneumococcal polysaccharides. In other aspects, a polysaccharide-protein conjugate formulation of the disclosure comprises one or more streptococcal polysaccharides. In yet other aspects, a polysaccharide-protein conjugate formulation of the disclosure comprises one or more meningococcal polysaccharides. In still other embodiments, a polysaccharide-protein conjugate formulation of the invention comprises a combination of one or more pneumococcal polysaccharides.
As defined hereinafter, the term "polysaccharide" is meant to include any antigenic saccharide element (or antigenic unit) commonly used in the immunologic and bacterial vaccine arts, including, but not limited to, a "saccharide", an "oligosaccharide", a "polysaccharide", a "liposaccharide", a "lipo-oligosaccharide (LOS)", a "lipopolysaccharide (LPS)", a "glycosylate", a "glycoconjugate" and the like.
In one particular embodiment of the invention, the one or more pneumococcal polysaccharides are a S. pneumoniae serotype 4 polysaccharide, a S. pneumoniae serotype 6B polysaccharide, a S. pneumoniae serotype 9V polysaccharide, a S. pneumoniae serotype 14 polysaccharide, a S. pneumoniae serotype 18C polysaccharide, a S. pneumoniae serotype 19F polysaccharide, a S. pneumoniae serotype 23F polysaccharide, a S. pneumoniae serotype 1 polysaccharide, a S. pneumoniae serotype 3 polysaccharide, a S. pneumoniae serotype 5 polysaccharide, a S. pneumoniae serotype 6A polysaccharide, a S. pneumoniae serotype 7F polysaccharide and a S. pneumoniae serotype 19A polysaccharide.
In certain embodiments, a polysaccharide-protein conjugate formulation is a 7-valent pneumococcal conjugate (7vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide.
In certain other embodiments, a polysaccharide-protein conjugate formulation is a 13-valent pneumococcal conjugate (13vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 1 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 3 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 5 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6A polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 7F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 19A polysaccharide conjugated to a CRM₁₉₇ polypeptide.
Polysaccharides are prepared by standard techniques known to those skilled in the art. For example, the capsular polysaccharides set forth in the present invention are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae, wherein each serotype is grown in a soy-based medium and the individual polysaccharides are then purified through centrifugation, precipitation, ultra-filtration, and column chromatography. The purified polysaccharides are then chemically activated (e.g., via reductive amination) to make the saccharides capable of reacting with the carrier protein. Once activated, each capsular polysaccharide is separately conjugated to a carrier protein (e.g., CRM₁₉₇) to form a glycoconjugate (or alternatively, each capsular polysaccharide is conjugated to the same carrier protein) and formulated into a single dosage formulation.
The chemical activation of the polysaccharides and subsequent conjugation to the carrier protein (i.e., a polysaccharide-protein conjugate) are achieved by conventional means. See, for example, U.S. Patent Nos. 4,673,574 and 4,902,506 .
Carrier proteins are preferably proteins that are non-toxic and non-reactogenic and obtainable in sufficient amount and purity. Carrier proteins should be amenable to standard conjugation procedures. In a particular embodiment of the present invention, CRM₁₉₇ is used as the carrier protein.
CRM₁₉₇ (Wyeth, Sanford, NC) is a non-toxic variant (i.e., toxoid) of diphtheria toxin isolated from cultures of Corynebacterium diphtheria strain C7 (β197) grown in casamino acids and yeast extract-based medium. CRM₁₉₇ is purified through ultra-filtration, ammonium sulfate precipitation, and ion-exchange chromatography. Alternatively, CRM₁₉₇ is prepared recombinantly in accordance with U.S. Patent No. 5,614,382 . Other diphtheria toxoids are also suitable for use as carrier proteins.
In other aspects, a carrier protein of the invention is an enzymatically inactive streptococcal C5a peptidase (SCP) (e.g., one or more of the SCP variants described in U.S. Patent 6,951,653 , U.S. Patent 6,355,255 and U.S. Patent 6,270,775 ).
Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (e.g., CT E29H, described in International Patent Application WO2004/083251 ), E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), or Haemophilus influenzae protein D, can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins.
After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugates are purified (enriched with respect to the amount of polysaccharide-protein conjugate) by a variety of techniques. These techniques include concentration/diafiltration operations, precipitation/elution, column chromatography, and depth filtration.
After the individual glycoconjugates are purified, they are compounded to formulate the immunogenic composition of the present invention. Formulation of the polysaccharide-protein conjugates of the present invention can be accomplished using art-recognized methods. For instance, the 13 individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
Particular aspects of the disclousre are set forth in the following numbered paragraphs:

1. A formulation which stabilizes a polysaccharide-protein conjugate, the formulation comprising (i) a pH buffered saline solution, wherein the buffer has a pKa of about 3.5 to about 7.5, (ii) a surfactant and (iii) one or more polysaccharide-protein conjugates.
2. The formulation of paragraph 1, wherein the polysaccharide-protein conjugate formulation is comprised in a container means selected from one or more of the group consisting of a vial, a vial stopper, a vial closure, a glass closure, a rubber closure, a plastic closure, a syringe, a syringe stopper, a syringe plunger, a flask, a beaker, a graduated cylinder, a fermentor, a bioreactor, tubing, a pipe, a bag, a jar, an ampoule, a cartridge and a disposable pen.
3. The formulation of paragraph 1, wherein the container means is siliconized.
4. The formulation of paragraph 1, wherein the buffer is succinate at a final concentration of 1 mM to 10 mM and pH 5.8 to 6.0.
5. The formulation of paragraph 1, wherein the surfactant is polysorbate 80.
6. The formulation of paragraph 5, wherein the final concentration of the polysorbate 80 in the formulation is at least 0.01% to 10% polysorbate 80 weight/volume of the formulation.
7. The formulation of paragraph 1, wherein the polysaccharide-protein conjugate comprises one or more pneumococcal polysaccharides.
8. The formulation of paragraph 1, further comprising one or more meningococcal polysaccharides, one or more meningococcal antigenic proteins, or a combination thereof.
9. The formulation of paragraph 1, further comprising one or more streptococcal polysaccharides, one or more streptococcal antigenic proteins, or a combination thereof.
10. The formulation of paragraph 1, wherein the polysaccharide-protein conjugate formulation is a 7-valent pneumococcal conjugate (7vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide.
11. The formulation of paragraph 1, wherein the polysaccharide-protein conjugate formulation is a 13-valent pneumococcal conjugate (13vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 1 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 3 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 5 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6A polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 7F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 19A polysaccharide conjugated to a CRM₁₉₇ polypeptide.
12. The formulation of paragraph 1, wherein the formulation further comprises an adjuvant.
13. The formulation of paragraph 12, wherein the adjuvant is aluminum phosphate.
14. A formulation which inhibits silicone induced aggregation of a polysaccharide-protein conjugate comprised in a siliconized container means, the formulation comprising (i) a pH buffered saline solution, wherein the buffer has a pKa of about 3.5 to about 7.5, (ii) an aluminum salt and (iii) one or more polysaccharide-protein conjugates.
15. The formulation of paragraph 14, wherein the buffer is succinate at a final concentration of 1 mM to 10 mM and pH 5.8 to 6.0.
16. The formulation of paragraph 14, further comprising polysorbate 80 (Tween™80), wherein the final concentration of the polysorbate 80 in the formulation is at least 0.01% to 10% polysorbate 80 weight/volume of the formulation.
17. The formulation of paragraph 14, wherein the polysaccharide-protein conjugate comprises one or more pneumococcal polysaccharides.
18. The formulation of paragraph 14, further comprising one or more meningococcal polysaccharides, one or more meningococcal antigenic proteins, or a combination thereof.
19. The formulation of paragraph 14, further comprising one or more streptococcal polysaccharides, one or more streptococcal antigenic proteins, or a combination thereof.
20. The formulation of paragraph 14, wherein the formulation further comprises an adjuvant.

E. EXAMPLES

The following examples are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail.

REFERENCE EXAMPLE 1

IMMUNOGENIC FORMULATIONS COMPRISING 0.001%-0.05% TWEEN™80 STABILIZE AND PREVENT AGGREGATION OF THE IMMUNOGEN

The polysaccharide-protein conjugate used in this example was a thirteen-valent pneumococcal polysaccharide conjugate (13vPnC) comprising capsular polysaccharides from S. pneumoniae serotypes 4, 6B, 9V, 18C, 19F, 14, 23F, 1, 3, 5, 6A, 7F and 19A, each of which were conjugated to CRM₁₉₇. The capsular polysaccharides were prepared by standard techniques known to those skilled in the art. Briefly, each pneumococcal polysaccharide serotype was grown in a soy-based medium, the individual polysaccharides were then purified through centrifugation, precipitation, ultra-filtration, and column chromatography. The purified polysaccharides were chemically activated for conjugation and each polysaccharide was separately conjugated to a CRM₁₉₇ carrier protein to form a glycoconjugate and formulated into a single dosage formulation.
The chemical activation of the polysaccharides and subsequent conjugation to the carrier protein were achieved by conventional means (e.g., see U.S. Patent No. 4,673,574 and 4,902,506 ). CRM₁₉₇ (Wyeth, Sanford, NC) is a non-toxic variant (i.e., toxoid) of diphtheria toxin isolated from cultures of Corynebacterium diphtheria strain C7 (β197) grown in casamino acids and yeast extract-based medium. CRM₁₉₇ is purified through ultra-filtration, ammonium sulfate precipitation, and ion-exchange chromatography.
The antigenicity experiments described below were performed by mixing the 13vPnC samples with one of thirteen antisera (Ab) specific to the each of the polysaccharide serotypes and detecting the immune complexes via light scattering measurements on an Array® 360 system (Beckman Coulter, Inc.; Fullerton, CA). The detected light scattering measurements for each of the thirteen serotypes were then compared to a standard curve and reported as antigenicity (µg/mL).
Syringes (BD Hypak SCF™) and syringe stoppers (BD Hypak SCF™) were purchased from BD Biosciences (Franklin Lakes, NJ). Clear borosilicate vials (VWR TraceClean™, 40 mL) with Teflon®-lined closures were purchased from VWR™ (West Chester, PA). Polysorbate 80 (Tween™80) was purchased from J.T. Baker (Mallinckrodt Baker, Inc.; Phillipsburg, NJ). Buffered saline was succinate (5 mM) and NaCl (0.85%) at pH 5.8.
The 13vPnC was formulated (500 mL total volume) at different surfactant concentrations (Tween™80; 0.001%, 0.005%, 0.01% and 0.05%, weight/volume) as follows: 0.85% saline (150 mM NaCl) was added to a one liter Pyrex® glass beaker, followed by 50 mM succinate buffer (final concentration 5 mM) and the 13vPnC. The final concentration of each serotype conjugate was 4.4 µg/mL (except for serotype 6B, which was 8.8 µg/mL). The 13vPnC formulation was then divided into five separate glass vials (50 mL per vial), wherein either 0.0%, 0.001%, 0.005%, 0.01% or 0.05% Tween™80 (w/v) was added to one of the five vials and each solution filtered through a 0.22 µm Durapore® filter (Millipore; Billerica, MA). Subsequently, 0.65 mL of each solution was filled in a separate 3 mL BD HYPAK™ SCF™ glass syringe with w4432 grey stoppers (BD Medical Pharmaceutical Systems; Franklin Lakes, NJ), and the syringes placed on a horizontal orbital shaker (60 cpm) for 100 hours at 2°C to 8°C.
It was observed by visual inspection (data not shown), that the13vPnC formulated in the absence of Tween™80 (i.e., 0.0%), would begin precipitating out of solution within ten minutes at 2-8°C upon gentle agitation via a horizontal orbital shaker. In contrast, the 13vPnC, formulated in 0.001%, 0.005%, 0.01% or 0.05% Tween™80 and gently agitated at 2-8°C, was stable for up to twenty-five days with no visible signs of precipitation (data not shown). Thus, this data demonstrated that the addition of a surfactant (e.g., Tween™80) to an immunogenic composition formulation enhances the stability of the immunogenic composition.
A second stability experiment of the 13vPnC further confirmed that the addition of surfactant to the formulation significantly enhanced the stability of the 13vPnC. In this experiment, the 13vPnC was formulated with and without 0.05% Tween™80. The 13vPnC formulated without Tween™80 (i.e., 0.0%) was prepared as follows: 0.85% saline (150 mM NaCl) was added to a one liter Pyrex® glass beaker, followed by 50 mM succinate buffer (final concentration 5 mM) and the 13vPnC, at a total volume of 500 mL. The 13vPnC formulation with 0.05% Tween™80 was prepared as follows: 0.85% saline (150 mM NaCl) was added to a one liter Pyrex® glass beaker, followed by 50 mM succinate buffer (final concentration 5 mM), 0.05% Tween™80 and the 13vPnC, at a total volume of 500 mL. The final concentration of each serotype conjugate in the 500 mL formulations was 4.4 µg/mL (except for serotype 6B, which was 8.8 µg/mL). The 500 mL formulations were homogenized via a rotor/stator homogenizer at 6,000 rpm (2-8°C) for 120 minutes. The homogenization process created an air-liquid interface (with air bubbles).
The stability of the 13vPnC formulation with (Table 1) and without (Table 1) 0.05% Tween™80 was assessed over a two hour time period as follows: Samples (20-30 mL) were removed at zero, thirty and one hundred-twenty minutes from the 0.0% and 0.05% Tween™80 formulations, the samples were diluted 1:2 in protein diluent (Array® 360 protein diluent (Cat. No. 663630); Beckman Coulter Inc.; Fullerton, CA) and the antigenicity of all thirteen serotypes of the 13vPnC were assayed (see, Table 1) on an Array® 360 system.

As is shown in Table 1, there was a significant decrease in antigenicity of the thirteen serotype polysaccharides (formulated without Tween™80) within the two hour assay. Quite significantly however, the 13vPnC formulation comprising 0.05% Tween™80 (Table 1), demonstrated robust stability with no reduction in the antigenicity throughout the two hour antigenicity assay.

TABLE 1

STABILITY ASSAY OF 13vPNC FORMULATED WITH AND WITHOUT TWEEN™80
13vPnC without Tween80				13vPnC with 0.05% Tween80
Serotype	Antigenicity 0 minutes	Antigenicity 30 minutes	Antigenicity 120 minutes	Serotype	Antigenicity 0 minutes	Antigenicity 30 min	Antigenicity 120 min
1	4.8 µg/ml	4.2 µg/ml	2.4 µg/ml	1	5.1 µg/ml	5.0 µg/ml	5.2 µg/ml
3	4.8 µg/ml	4.1 µg/ml	1.7 µg/ml	3	5.0 µg/ml	5.0 µg/ml	5.2 µg/ml
4	5.8 µg/ml	5.0 µg/ml	3.1 µg/ml	4	6.1 µg/ml	6.1 µg/ml	6.2 µg/ml
5	3.4 µg/ml	3.0 µg/ml	2.0 µg/ml	5	3.6 µg/ml	3.6 µg/ml	3.7 µg/ml
6A	4.9 µg/ml	3.8 µg/ml	1.3 µg/ml	6A	5.4 µg/ml	5.4 µg/ml	5.6 µg/ml
6B	10.0 µg/ml	5.6 µg/ml	1.4 µg/ml	6B	10.6 µg/ml	10.6 µg/ml	10.5 µg/ml
7F	4.7 µg/ml	3.4 µg/ml	1.0 µg/ml	7F	5.3 µg/ml	5.2 µg/ml	5.3 µg/ml
9V	5.6 µg/ml	4.7 µg/ml	2.5 µg/ml	9V	6.1 µg/ml	6.1 µg/ml	6.2 µg/ml
14	7.6 µg/ml	6.4 µg/ml	3.0 µg/ml	14	8.2 µg/ml	8.3 µg/ml	8.3 µg/ml
18C	5.6 µg/ml	4.4 µg/ml	1.7 µg/ml	18C	6.2 µg/ml	6.1 µg/ml	6.2 µg/ml
19A	6.4 µg/ml	4.5 µg/ml	1.9 µg/ml	19A	6.8 µg/ml	6.8 µg/ml	6.8 µg/ml
19F	5.4 µg/ml	2.6 µg/ml	0.0 µg/ml	19F	6.1 µg/ml	6.2 µg/ml	6.0 µg/ml
23F	4.5 µg/ml	2.8 µg/ml	0.9 µg/ml	23F	5.2 µg/ml	5.2 µg/ml	5.2 µg/ml

The 13vPnC/Tween™80 formulation was further tested for stability against high shear forces. In this experiment, a 100 mL 13vPnC composition (4.4 µg/mL serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F and 8.8 µg/mL serotype 6B, 5 mM succinate buffer, 150 mM NaCl and 0.25 mg/mL AlPO₄) was added to three 250 mL glass bottles comprising either 0.0%, 0.01% or 0.05% Tween™80. The three bottles were then vortexed for thirty minutes (2-8°C) on a vortexer (Vortex-Genie® 2; Scientific Industries, Inc.; Bohemia, NY) and an air-liquid interface was created at the maximum speed setting. After thirty minutes 10-30 mL samples were taken from each bottle, diluted 1:2 in Array® 360 protein diluent and the antigenicity of the thirteen serotypes assayed on an Array® 360 system.

As seen in Table 2 below, the 13vPnC formulated without Tween™80 (0.0%) had on average a 20% decrease in antigenicity after vortexing. The 13vPnC formulated with 0.01% Tween™80 had a decrease in antigenicity ranging from 2-10% (average 8%) and the 13vPnC formulated with 0.05% Tween™80 had a decrease in antigenicity ranging from 0-8% (average 3%). Thus, the data presented in Table 2 demonstrate that the 13vPnC formulated with either 0.01% or 0.05% Tween™80 were significantly stabilized against shear forces, relative to the 13vPnC formulated in the absence of Tween™80.

TABLE 2

STABILIZING EFFECT OF TWEEN™80 AGAINST SHEAR FORCES
Serotype	Antigenicity 0.0% tw80	Antigenicity 0.0% tw80 + vortex	Antigenicity 0.01% tw80	Antigenicity 0.01% tw80 + vortex	Antigenicity 0.05% tw80	Antigenicity 0.05% tw80 + vortex
1	4.7 µg/mL	3.6 µg/mL	4.8 µg/mL	4.3 µg/mL	4.7 µg/mL	4.6 µg/mL
3	4.6 µg/mL	3.4 µg/mL	4.7 µg/mL	4.2 µg/mL	4.7 µg/mL	4.4 µg/mL
4	5.5 µg/mL	4.4 µg/mL	5.9 µg/mL	5.4 µg/mL	5.9 µg/mL	5.6 µg/mL
5	3.2 µg/mL	2.5 µg/mL	3.5 µg/mL	3.2 µg/mL	3.3 µg/mL	3.3 µg/mL
6A	4.3 µg/mL	3.6 µg/mL	4.6 µg/mL	4.5 µg/mL	4.7 µg/mL	4.8 µg/mL
6B	9.7 µg/mL	7.7 µg/mL	10.2 µg/mL	9.6 µg/mL	10.2 µg/mL	10.1 µg/mL
7F	4.6 µg/mL	3.5 µg/mL	5.4 µg/mL	5.0 µg/mL	5.4 µg/mL	5.3 µg/mL
9V	5.3 µg/mL	4.1 µg/mL	5.7 µg/mL	5.1 µg/mL	5.6 µg/mL	5.3 µg/mL
14	6.8 µg/mL	5.4 µg/mL	7.3 µg/mL	6.7 µg/mL	7.4 µg/mL	6.8 µg/mL
18C	4.1 µg/mL	3.4 µg/mL	4.5 µg/mL	4.3 µg/mL	4.5 µg/mL	4.5 µg/mL
19A	5.1 µg/mL	4.2 µg/mL	5.5 µg/mL	5.3 µg/mL	5.6 µg/mL	5.4 µg/mL
19F	4.8 µg/mL	3.6 µg/mL	5.2 µg/mL	4.9 µg/mL	5.2 µg/mL	5.1 µg/mL
23F	3.0 µg/mL	2.4 µg/mL	3.4 µg/mL	3.3 µg/mL	3.5 µg/mL	3.4 µg/mL

REFERENCE EXAMPLE 2

FORMULATIONS COMPRISING SURFACTANT STABILIZE AND PREVENT AGGREGATION OF STREPTOCOCCAL C5A PEPTIDASE

The streptococcal C5a peptidase (SCP) used in this example was expressed and purified as follows. The SCP was expressed recombinantly in E. coli using an arabinose inducible system. Standard fermentation protocols for E. coli using animal-free defined medium and subsequent cell lysis were followed. Recombinant SCP was purified from the soluble fraction of the cell lysate by saturation to 60% (approximately 3 M) ammonium sulfate while stirring for 12-24 hours. The saturated lysate was centrifuged, supernatant retained and loaded onto a phenyl Sepharose hydrophobic interaction column. Bound material was then eluted with 1 M ammonium sulfate, 20mM Tris-CI, pH 7.5, concentrated, and diafiltered against PBS, pH 7.4. The purified recombinant SCP (rSCP) was diluted to -10 mg/mL with PBS, pH 7.4 and passed through a Posidyne filter to remove endotoxin, followed by a final filtration (0.2 mM) for sterility and stored frozen (-25°C).
The purified SCP (55 µg/mL) was then formulated with 0.025% Tween™80 or without Tween™80 (0.0%) in the following buffers: 5 mM succinate buffer at pH 6.0, 10 mM phosphate buffer at pH 7.0, 10 mM phosphate buffer at 7.4 or 10 mM Tris buffer at pH 7.5 and filled in separate BD Hypak SCF™ syringes. The syringes were then placed on an a horizontal orbital shaker at 2-8°C, shaken at 180 cpm for two days and the SCP protein concentration determined by the modified Lowry assay.
As shown in FIG. 1, the stability of SCP was greatly enhanced when formulated with Tween™80. For example, after two days on the orbital shaker, the SCP formulated without Tween™80 (FIG. 1A) demonstrated a significant decrease (e.g., greater than 90%) in the SCP concentration each of the buffers tested. However, as shown in FIG. 1B, the addition of 0.025% Tween™80 to the SCP buffer formulations, prior to being placed on the orbital shaker for two days, completely inhibited the SCP loss which was observed in FIG. 1A.
The storage stability of the SCP/Tween™80 (0.025%) formulation was also assessed at 25°C and 37°C for eight weeks and six weeks, respectively (data not shown). Briefly, the SCP (200 µg) was formulated in either succinate buffer or phosphate buffer as follows: succinate buffer (5 mM, pH 6.0) or phosphate buffer (15 mM, pH 7.4), 0.9% NaCl and 0.025% Tween™80. The stability of the SCP/Tween™80 formulations were assayed by size-exclusion-HPLC, modified Lowry total protein assay and visual inspection for precipitation. It was observed in this study, that the SCP/Tween™80 formulations (in either buffer) were completely stable at 25°C and 37°C for the entire stability study (i.e., up to eight weeks and six weeks, respectively).

EXAMPLE 3

THE INFLUENCE OF SILICONIZED CONTAINER MEANS ON THE STABILITY OF 13VPNC

Previous experiments indicated (data not shown) that 13vPnC immunogenic compositions precipitated and/or aggregated when filled in ready to use (single-dose) Becton Dickinson® (BD) Hypak Type 1 borosilicate glass syringes treated with Dow Corning® medical grade DC 360 silicone and capped with West 4432/50 latex free stoppers (chlorobutyl) and EZ tip cap West 7025/65 (Synthetic Isoprene Bromobutyl Blend; West Pharmaceutical®, Lionville, PA). In these experiments, the 13vPnC was formulated in 5 mM succinate buffer containing 0.85% NaCl and 4.4 µg/ml of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F and 8.8 µg/ml of S. pneumoniae serotype 6B, with and without 0.25mg/mL aluminum phosphate as an adjuvant. It was observed that, in the absence of AlPO₄, the 13vPnC particulates were readily observable, whereas, in the presence of AlPO₄, the 13vPnC particulates were significantly diminished and more difficult to detect.
In the present example, a series of container and closure components (i.e., container means) were examined to identify what components were inducing or contributing to 13vPnC particulate formation. The container means tested comprised syringes, stoppers and vials and are listed below in Table 3. The BD and West stoppers listed in Table 3 were siliconized, using either the Huber or Jar process. The Huber process of siliconization is more controlled and yielded 30 to 60 µg/cm2 of silicone on the surface of the stopper, while the Jar process of siliconization resulted in 150 to 300 µg/cm2 of silicone on the surface of the stopper. Based on theoretical calculations, about 15% of the surface area of the stopper is exposed to the product in the syringe, suggesting that for the Huber and Jar process between 4.5 to 9 µg and 22.5 to 45 µg of silicone is extractable from the stoppers, respectively.

Materials

The silicone was Dow Corning® 360 Medical Fluid 1000 centistokes (batch No. 0001846266). The 7vPnC was formulated in 5 mM succinate buffer containing 0.85% NaCl and 4.4 µg/ml of S. pneumoniae serotypes 4, 9, 14, 18C, 19F and 23F and 8.8 µg/ml of S. pneumoniae serotype 6B, with and without 0.25 mg/ml aluminum phosphate. The 13vPnC was formulated in 5 mM succinate buffer containing 0.85% NaCl and 4.4 µg/ml of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F and 8.8 µg/ml of S. pneumoniae serotype 6B, with and without 0.25 mg/ml aluminum phosphate. Monovalent S. pneumoniae serotype 6B was formulated (5 mM succinate buffer containing 0.85% NaCl, without aluminum phosphate) at a concentration of 61 µg/ml to simulate the total saccharide concentration of the 13vPnC formulations.

Methods

The 7vPnC and 13vPnC were formulated as described above, and 35 ml of a given formulation was added to a clear 250 ml Nalgene® bottle. Into each Nalgene® bottle, the container means components listed in Table 3 were added. The Nalgene® bottles were then placed on a Labline® Orbit Shaker and swirled overnight at 50 rpm. The results are summarized in Table 3.
Visual Appearance. The Nalgene® bottles containing each of the container means components were held up to a fluorescence light in the laboratory. A path of a beam of light (Tindel effect) passing through the samples allowed for the detection of particulates.
Protein Assay. The total protein and protein bound to aluminum was determined by measuring the total protein concentration in the formulated immunogenic composition and the protein associated with the aluminum pellet, respectively (an aliquot of the immunogenic composition was centrifuged and the pellet was re-suspended in saline). Assays were performed using the Pierce Modified Lowry protein assay (catalog # 23240) with bovine serum albumin as a standard.

RESULTS

In the first series of experiments, the 13vPnC immunogenic compositions were formulated without AlPO₄ and exposed to a series of container means listed below in Table 3. It was clearly evident from the data (Table 3), that the container means components that were treated with silicone oil induced the formation of white particles. In contrast, no particulates were detected in the presence of the non-siliconized Daikyo® stoppers (Daikyo Seiko, Ltd., Japan) and Schott vials (Schott North America Inc.; Lebanon, PA).

TABLE 3

EFFECT OF DIFFERENT CONTAINER MEANS COMPONENTS ON 13vPNC, FORMULATED WITHOUT ALPO₄
Container Means Components	Number of Container Means Components Added	Appearance (Visual Inspection)
Control-13vPnC without AlPO4	None	No Particulate
BD Hypak BSCF 1-3 ml 4432/50 Grey Si WWD Stoppers	10	Particulates
BD Hypak BSCF 1-3 ml 4432/50 Grey Si Huber Processed Stoppers	10	Particulates
West 890 Ready to Sterilize Stoppers	10	Particulates
BD Hypak BSCF 1-3 ml W4416/50 Grey Si 1000 WWD Stoppers	10	Particulates
Helvoet 6213 Stoppers	10	Particulates
Daikyo Vial Stoppers (D777-1 B2-40 F451 plug stoppers)	10	No Particulate
BD Hypak BSCF 1-3ml LLA EZGTC W7025/65 Syringe Barrels	4	Particulates
Hypak NSCF 1-3 ml 4023/50 B2-40 Daikyo Stoppers	10	No Particulate
Syringe E-Z Grip Tip Cap W7025/65 EZ IITC	10	No Particulate
2 ml, 13 mm Schott Type 1 glass vials	4	No Particulate
Silicone Oil (Dow Chemical Medical grade 360)	500 µL (1.43%)	Particulates
Schott TopPac Syringes	4	No Particulate

The monovalent S. pneumoniae serotype 6B was chosen as a model for the 13vPnC and was formulated at 61.6 µg/ml (without AlPO₄) to simulate the total saccharide concentration in the 13vPnC formulation. Silicone (Dow Corning 360 Medical Fluid) was added to aliquots of the formulated monovalent 6B, ranging from 2 ppm to 100 ppm. The mixtures were placed on a Labline® Orbit Shaker for 2 hours at 50 rpm. As indicated below in Table 4, fiber-like white particulates were observed at all silicone (Si) concentrations.

TABLE 4

EFFECT OF SILICONE CONCENTRATION ON THE FORMATION OF PARTICULATES
Silicone Concentration	Appearance (Visual Inspection)
2 ppm (1 µl of Si to 500 mL Formulation)	Fiber-like white particulates
5 ppm (2.5 µl of Si to 500 mL Formulation)	Fiber-like white particulates
10 ppm (5 µl of Si to 500 mL Formulation)	Fiber-like white particulates
15 ppm (7.5 µl of Si to 500 mL Formulation)	Fiber-like white particulates
20 ppm (10 µl of Si to 500 mL Formulation)	Fiber-like white particulates
100 ppm (2 µl of Si to 20 mL Formulation)	Fiber-like white particulates

The amount of silicone in 13vPnC formulations (without AlPO₄) was also examined. The silicone concentration was determined by DC Plasma Emission Spectroscopy (data not shown). In this method, the content of 25 syringes were pooled and extracted with two 50 ml portions of cyclohexane/isopropyl alcohol mixture. The extracts were combined and evaporated. The residual was solubilized and tested as per existing methods for silicone determination on rubber stoppers. The results indicated that between 15.8 to 19.0 µg of silicone is extractable from each syringe. This amount corresponds to 2.7% to 3.3% of silicone.
In a separate series of experiments, in which the 13vPnC was formulated in the presence of AlPO₄ and subjected to the same container means set forth in Table 3, it was elucidated that the silicone and the "free" protein (13vPnC) in solution was responsible for the formation of the particulates (data not shown). FTIR analysis of the particulates also indicated that the particulate consisted of protein and silicone (data not shown). It was determined in these experiments, that about 85% of the 13vPnC is bound to the AlPO₄, wherein the remaining 15% was free (not bound to AlPO₄) 13vPnC in solution. In contrast, it was observed that 7vPnC formulated with AlPO₄ was 100% bound to the AlPO₄ (data not shown).
To elucidate the effect of free protein-polysaccharide on the formation of particulates, 25 ml of both 7vPnC and 13vPnC were aliquoted and transferred to a 50 ml centrifuge tube. The samples were centrifuged for 10 minutes at 3,000 rpm and the supernatant was carefully extracted and transferred to a Nalgene® bottle. Ten siliconized stoppers (4432 Stoppers) were added to each bottle and placed on orbital shaker at 50 rpm. After careful visual inspection, it was observed that the 7vPnC supernatant exhibited no particulate formation, thereby remaining clear and colorless. However, the 13vPnC supernatant began to show low levels of particulate in the fourth hour of observation (data not shown). This result suggested that the free protein-polysaccharide in solution, in conjunction with silicone, is responsible for the formation of the particulates.

To further elucidate the contribution of the free protein-polysaccharide in solution to the formation of particulates, monovalent S. pneumoniae serotypes 4 and 6B were chosen for their high and low binding to aluminum, respectively. These two monovalents were formulated at protein concentration ranging from 25 µg/ml to 200 µg/ml in the absence and presence of AlPO₄. Ten siliconized stoppers (4432 stoppers) were placed in each formulation, which were then placed on the orbit shaker at 50 rpm. As indicated below in Table 5, fiber-like white particulates were observed for both monovalent serotypes at all protein concentrations in the absence of AlPO₄. However, in the presence of AlPO₄, particulates were detected at lower concentrations for serotype 4 (100 µg/ml) versus serotype 6B (200 µg/ml), data not shown.

TABLE 5

EFFECT OF PROTEIN CONCENTRATION ON THE FORMATION OF PARTICULATES
	Appearance (Visual Inspection)
	Without AlPO₄	With AlPO₄
25 µg/mL of 6B	Fiber-like white particulates	No particulates
50 µg/mL of 6B	Fiber-like white particulates	No particulates
75 µg/mL of 6B	Fiber-like white particulates	No particulates
100 µg/mL of 6B	Fiber-like white particulates	No particulates
200 µg/mL of 6B	Fiber-like white particulates	Fiber-like white particulates
25 µg/mL of Type 4	Fiber-like white particulates	No particulates
50 µg/mL of Type 4	Fiber-like white particulates	No particulates
75 µg/mL of Type 4	Fiber-like white particulates	No particulates
100 µg/mL of Type 4	Fiber-like white particulates	Fiber-like white particulates
200 µg/mL of Type 4	Fiber-like white particulates	Fiber-like white particulates

EXAMPLE 4

ALUMINUM ADJUVANTS INHIBIT THE FORMATION OF 13VPNC PARTICULATES IN THE PRESENCE OF SILICONIZED CONTAINER MEANS

As set forth above in Example 3, a 13vPnC immunogenic composition is a liquid formulation comprising 4.4 µg/mL of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F and 8.8 µg/mL of type 6B in 5 mM succinate buffer (pH 5.8) and 0.85% NaCl, which may also be formulated with or without an adjuvant (e.g., an aluminum adjuvant). The 13vPnC may also be formulated with or without an adjuvant, such as 0.25 mg aluminum/ml aluminum phosphate (AlPO₄). It was observed in Example 3, that 13vPnC formulated without AlPO₄ and filled in BD Hypak SCF™ syringes (capped with Hypak plungers) failed visual inspection due to the observation of particulates, wherein further studies revealed that the particulates were in part a result of protein-polysaccharide interactions with silicone. In the following example, syringes (and plungers) from various vendors were evaluated with 13vPnC formulations, wherein shipping and handling conditions were simulated via agitation (described below).

Materials

The 13vPnC was formulated in 5 mM succinate buffer containing 0.85% NaCl and 4.4 µg/ml of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F and 8.8 µg/ml of S. pneumoniae serotype 6B, with and without 0.25 mg/ml aluminum phosphate. The container means tested are listed below in Table 6.

TABLE 6

CONTAINER MEANS
		Container Means	Description
	1	Vetter syringes Type 1 untreated glass	1 ml long bulk format
	2	Schott TopPac® syringes	Plastic syringes
	3	BD Baked syringes Type 1 untreated glass	0.1 mg silicone/barrel
	4	BD Baked syringes Type 1 untreated glass	0.04 mg silicone/barrel
	5	BD High viscosity syringes	2.25 ml syringes
		Type
1 untreated glass	12500 cst silicone
	6	BD High viscosity syringes	1.0 ml syringes
		Type
1 untreated glass	12500 cst silicone
	7	BünderGlas syringes, PS2 Type 1 untreated glass	0.056 mg silicone/barrel
	8	BünderGlas syringes, PS4 Type 1 untreated glass	0.14 mg silicone/barrel
	1	West 4023/50 Flurotec® B2-40 plungers	Flurotec® plungers
	2	West 4023/50 Flurotec® B2-40 plungers	Flurotec® plungers
	1	13vPnC with AlPO₄ in BD Hypak syringes with West 4432 ready to use plungers and 7025/65 EZ tip caps	Positive control, high silicone
	2	13vPnC with AlPO₄ in un-siliconized syringes with West 4023/50 Flurotec® B2-40 plungers	Negative control, not treated with silicone

Methods

Formulation and Fill Procedure. Listed below in Table 7 is the recipe for a 2 liter 13vPnC formulation. Briefly, the 0.85 % saline was first added to a glass beaker, followed by the 5 mM succinate buffer (pH 5.8), and then sequentially each of the S. pneumoniae serotype conjugates. The formulation was then gently mixed on a stirrer plate and filtered through a 0.22 µm Millipore® filter unit. For formulation comprising AlPO₄ , the AlPO₄ (0.25 mg/ml final concentration) was then added and the formulation gently mixed. The test syringes were then filled (0.58 ml/syringe) and capped with plungers.
Shipping Simulation via Agitation. A VWR® signature Digital Multitube vortexer (Catalog No. 14005-826) was used to agitate the samples. The syringes filled with 13vPnC were placed horizontal and fixed by the two support plates of the vortexer. Samples were held at horizontal position and agitated at 500 rpm pause mode at 2-8°C for twenty-four hours.

Nephelometry. Serotype specific antigenicities were determined by a rate nephelometry assay using type-specific antibodies. For 13vPnC with AlPO₄, the aluminum phosphate was solubilized by adding 1N NaOH. The solution was immediately neutralized by adding 1M citric acid. For 13vPnC without AlPO4, no solubilization and neutralization procedures were performed. The assay measures the rate of change of light scattering intensity derived from the antibody-antigen complex formed in the sample using Beckman Array 360 nephelometer.

TABLE 7

13vPNC FORMULATION TABLE
Component	Batch Size (L)	Bulk Conc (mg/mL)	Required Conc (ug/mL)	13vPnC with AlPO₄ Volume (mL)	13vPnC without AlPO₄ Volume (mL)
serotype 1	2.000	0.506	4.4	17.39	17.39
serotype 3	2.000	0.256	4.4	34.38	34.38
serotype 4	2.000	0.530	4.4	16.60	16.60
serotype 5	2.000	0.515	4.4	17.09	17.09
serotype 6A	2.000	0.519	4.4	16.96	16.96
serotype 6B	2.000	0.489	8.8	35.99	35.99
serotype 7F	2.000	0.500	4.4	17.60	17.60
serotype 9V	2.000	0.521	4.4	16.89	16.89
serotype 14	2.000	0.518	4.4	16.99	16.99
serotype 18C	2.000	0.509	4.4	17.29	17.29
serotype 19A	2.000	0.511	4.4	17.22	17.22
serotype 19F	2.000	0.520	4.4	16.92	16.92
serotype 23F	2.000	0.511	4.4	17.22	17.22
Succinate Buffer in 0.85% Saline, pH 5.8	2.000	50.0	5000	200.0	200.0
AlPO₄	2.000	3.250	250	153.85	NA
Saline	2.000	NA	NA	1387.62	1541.46

RESULTS

In this study, syringes from different venders, having different silicone levels (Table 6), were subject to controlled agitation conditions. The total antigenicity of each serotype was measured by Nephelometry assay for both pre-agitation and post-agitation samples. Antigenicity loss following agitation (percentage) was calculated and is shown in FIG. 2 through FIG. 7.
Prior to the study, the agitation conditions were optimized based on the antigenicity loss of the two controls: (1) the worst-case control (positive control, high silicone; FIG. 2) and (2) the best-case control (negative control, no silicone; FIG. 3). The conditions were then optimized such that the antigenicity loss was low in positive control, yet detectable in the negative control. This was to ensure that the agitation was neither too weak to produce precipitation in the syringes; nor too strong, such that the precipitation might be caused by factors other than the silicone interaction (e.g., by shear forces). Thus, agitation at 500 rpm (pause mode) for twenty-four hours was chosen as the most suitable agitation condition, while a temperature of 2-8°C and a horizontal position were used to simulate the conditions in real time product shipping and handling.
The results of the study are summarized as follows: The largest antigenicity losses of the 13vPnC formulated with AlPO₄ occurred in the syringes with higher silicone levels (data not shown). For example, of the syringes listed in Table 6, the BD Hypak syringe (control 1), the BD baked syringe (syringe 3; 0.1 mg silicone), the BD high viscosity (syringe 5) and the BünderGlas PS4 syringe (syringe 8, 0.14 mg silicone), each had one or more of the 13vPnC serotypes with greater than 10% antigenicity loss. The smallest antigenicity losses of the 13vPnC formulated with AlPO₄ occurred in the syringes with lower silicone levels. For example, the Vetter syringes (FIG. 4) and the Schott TopPac plastic syringes (FIG. 5) were most similar to un-siliconized syringes (FIG. 2), both demonstrating minor antigenicity losses for 13vPnC formulated with AlPO₄.
The influence of the aluminum phosphate on stabilizing the 13vPnC and inhibiting particulate formation in the presence of siliconized syringes was analyzed in experiments using 13vPnC formulated with and without 0.25 mg/ml AlPO₄, wherein syringes used were the BD baked low silicone syringes (syringe 4 in Table 6) and the BünderGlas low silicone PS2 syringes (syringe 7 in Table 6). The BD baked low silicone syringes (0.04 mg silicone/barrel) typically had less than 10% antigenicity loss for the 13vPnC serotypes formulated with AlPO₄ (FIG. 6A), whereas the antigenicity loss for the 13vPnC serotypes formulated without AlPO₄ (FIG. 6B) had antigenicity losses ranging from 5% (serotype 1) up to about 50% (serotype 23F). The BünderGlas low silicone PS2 (0.056 mg silicone/barrel) syringes had less than 5-8% antigenicity loss (depending on serotype) for 13vPnC formulated with AlPO₄ (FIG. 7A), whereas the antigenicity loss for the 13vPnC serotypes formulated without AlPO₄ (FIG. 7B) had antigenicity losses ranging from about 5% to about 30% (depending on serotype).
Thus, these data taken together indicate that: (1) the antigenicity loss of 13vPnC was greater in the syringes with higher silicone levels and (2) the 13vPnC formulated without AlPO₄ sustained greater antigenicity losses than 13vPnC with AlPO₄ in all of the syringes tested.

REFERENCE EXAMPLE 5

FORMULATIONS COMPRISING SURFACTANT OPTIMIZE THE BINDING OF MENINGOCOCCAL ANTIGENIC PROTEINS TO ALUMINUM SALT ADJUVANTS

The recombinant lipidated N. meningitidis 2086 protein (rLP2086) used in this example was expressed and purified as follows. The rLP2086 was expressed recombinantly in E. coli utilizing a native leader sequence. Standard fermentation protocols for E. coli using animal-free defined medium and subsequent cell lysis were followed. Recombinant lipidated N. meningitidis 2086 protein was purified from the membrane pellet with 50mM Tris-HCI/5mM EDTA/1% sarcosyl pH 8. This sarcosyl extract was adjusted to 1 % Zwittergent 3-14 (Z3-14) and dialyzed twice against a 30 fold excess of 50mM Tris-HCI/5mM EDTA/1% Z3-14. The dialyzed rLP2086 extract was precipitated with 90% ethanol to remove remaining sarcosyl, and solubilized with 50mM Tris-HCI/5mM EDTA/1% Z3-14 pH 8. Insoluble material was removed by centrifugation, the supernatant was passed over an anion exchange chromatography column, and rLP2086 was collected in the unbound fraction. The unbound material was then dialyzed twice against a 30-fold excess of 25mM NaAc/1% Z3-14 pH 4.5, and passed over a cation exchange chromatography column. The rLP2086 was eluted with a 0-0.3M NaCl gradient and stored frozen (-25°C).

The purified rLP2086 was then formulated with 150 mM NaCl, 0.020% Tween™80, 0.25 mg Al/mL of AlPO₄, and in the following buffers: 10 mM phosphate buffer at pH 7.0 and 5 mM succinate buffer at pH 6.0. Table 8 compares protein binding percentage to the AlPO₄ adjuvant.

TABLE 8

RLP2086 BINDING TO ADJUVANT
Buffer	Total Protein Conc. (µg/mL)	AlPO₄ Bound Protein (%)
10 mM Phosphate buffer pH 7.0 containing 150 mM NaCl, 0.02% polysorbate 80 and 0.25 mg Al/mL of AlPO₄	400	68
	120	82
5 mM Succinate buffer pH 6.0 containing 150 mM NaCl, 0.02% polysorbate 80 and 0.25 mg Al/mL of AlPO₄	400	81
	120	100

REFERENCES

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Claims

A siliconized container means filled with a formulation which inhibits silicone induced aggregation of a polysaccharide- protein conjugate comprised in a siliconized container means, the formulation comprising (i) a pH buffered saline solution, wherein the buffer has a pKa of about 3.5 to about 7.5, (ii) an aluminum salt and (iii) one or more polysaccharide-protein conjugates wherein the polysaccharide-protein conjugate comprises one or more pneumococcal polysaccharides.
The siliconized container means of claim 1, wherein the pH buffered saline solution in the formulation has a pH of 5.5 to 7.5.
The siliconized container means of claim 1 or 2, wherein the buffer in the formulation is phosphate, succinate, histidine or citrate.
The siliconized container means of claim 1 wherein the buffer is succinate at a final concentration of 1 mM to 10 mM and pH 5.8 to 6.0.
The siliconized container means of anyone of claims 1 to 4, wherein the salt in the pH buffered saline solution comprises magnesium chloride, potassium chloride, sodium chloride or a combination thereof.
The siliconized container means of anyone of claims 1 to 5, wherien the aluminum salt is aluminum hydroxide, aluminum phosphate or aluminum sulfate.
The siliconized container means of anyone of claims 1 to 5 wherein the aluminum salt is aluminum phosphate.
The siliconized container means of anyone of claims 1 to 7 wherein the formulation further comprises polysorbate 80 (Tween™80).
The siliconized container means of claim 8 wherein the final concentration of the polysorbate 80 in formulation is at least 0.01% to 10% polysorbate 80 weight/volume of the formulation.
The siliconized container means of anyone of claims 1 to 9, wherein the one or more pneumococcal polysaccharides are a S. pneumoniae serotype 4 polysaccharide, a S. pneumoniae serotype 6B polysaccharide, a S. pneumoniae serotype 9V polysaccharide, a S. pneumoniae serotype 14 polysaccharide, a S. pneumoniae serotype 18C polysaccharide, a S. pneumoniae serotype 19F polysaccharide, a S. pneumoniae serotype 23F polysaccharide, a S. pneumoniae serotype 1 polysaccharide, a S. pneumoniae serotype 3 polysaccharide, a S. pneumoniae serotype 5 polysaccharide, a S. pneumoniae serotype 6A polysaccharide, a S. pneumoniae serotype 7F polysaccharide and a S. pneumoniae serotype 19A polysaccharide.
The siliconized container means of anyone of claims 1 to 10, wherein the protein of the polysaccharide-protein conjugate formulation is selected from the group consisting of CRM₁₉₇, a tetanus toxoid, a cholera toxoid, a pertussis toxoid, an E. coli heat labile toxoid (LT),a pneumolysin toxoid, pneumococcal surface protein A (PspA), pneumococcal adhesin protein A (PsaA), a C5a peptidase from Streptococcus, Haemophilus influenzae protein D, ovalbumin, keyhole limpet haemocyanin (KLH), bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD).
The siliconized container means of anyone of claims 1 to 9, wherein the polysaccharide-protein conjugate formulation is a 7-valent pneumococcal conjugate (7vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide.
The siliconized container means of anyone of claims 1 to 9, wherein the polysaccharide-protein conjugate formulation is a 13-valent pneumococcal conjugate (13vPnC) formulation comprising a S. pneumoniae serotype 4 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6B polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 9V polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 14 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 18C polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 19F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 23F polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 1 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 3 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 5 polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 6A polysaccharide conjugated to a CRM₁₉₇ polypeptide, a S. pneumoniae serotype 7F polysaccharide conjugated to a CRM₁₉₇ polypeptide and a S. pneumoniae serotype 19A polysaccharide conjugated to a CRM₁₉₇ polypeptide.
The siliconized container means of any one of claims 1 to 13 wherein said container is a syringe.
The siliconized container means of claim 14 wherein said container is a glass syringe.